Urothelium synthesizes several integral membrane proteins called uroplakins as its major differentiation products. These proteins form 16-nm particles packed hexagonally in two dimensional crystals of urothelial plaques which cover greater than 90 percent of the apical urothelial surface. The long-term goal of this project is to elucidate the molecular structure, biological function and disease implications of uroplakins and several other urothelial plaque proteins. Three specific aims will be pursued during the next five years. First, studies will be conducted to characterize two novel urothelial plaque proteins. p35 is a urothelial specific protein sharing sequence homologies with both uroplakin III and PMSR6, a DNA mismatch repair enzyme (MMR)-related protein. As mutations in some MMR proteins are known to cause hereditary nonpolypoid colorectal cancer, it is intriguing why a urothelial plaque protein contains a PMSR6-like sequence in its extracellular domain. p18 is a major urothelial plaque protein that has been ignored previously because it is only partially resolved from uroplakin II by SDS- PAGE and because it does not transfer well electrophoretically. Studies will be conducted to define the structure and function of these two novel urothelial markers. Second bifunctional chemical crosslinking reagents will be used to crosslink the neighboring uroplakins, and the amino acid sequences of the abutting, crosslinked subdomains of the crosslinked uroplakins will be determined by microsequencing using mass spectrometry. The data will define the neighboring relationships between individual uroplakins in terms of subdomain interactions. Finally, genes encoding several urothelial differentiation-related proteins including uroplakins Ia, Ib and p35 will be ablated or mutated to generate mice that are deficient or defective in these urothelial-specific genes. The phenotype of such mutant mice will be characterized to determine whether they provide novel animal models and whether they give clues to the pathophysiology of human urological diseases. These studies are important, as they will complete the characterization of all the major urothelial plaque-associated proteins; they will tell us how the four uroplakins interact to form the amazingly beautiful urothelial plaque structure; they will reveal the in vivo, biological functions of uroplakin I's and p35; and they will tell us whether ablation or specific mutations of these major urothelial markers can cause different subtypes of vesicoureteral reflux and possibly other human urological diseases.